A near-infrared squaricine dye with ultra-high fluorescence brightness, its preparation method and application

Abstract

This invention belongs to the fields of functional fluorescent dyes and biomedical technology, specifically relating to a near-infrared squaric acid cyanine dye with ultra-high fluorescence brightness, its preparation method, and its application. A water-soluble charged 1,8-naphthoylimide derivative is obtained through an imino substitution reaction of 1,8-naphthoylimide, which is then coupled with squaric acid to obtain a near-infrared squaric acid cyanine dye with ultra-high fluorescence brightness. The obtained near-infrared squaric acid cyanine dye is then reacted with mPEG. 2k Nanoparticles were obtained by co-assembling DSPE ammonium salt. Compared to the dye in aqueous solution, the dye in the co-assembled nanoparticles exhibited a 58-fold increase in fluorescence intensity, which is attributed to the charge and squaric acid parent compound in the molecular structure, as well as mPEG. 2k The electrostatic interaction of DSPE ammonium salt is related to the reduction of molecular vibrations, which gives the resulting nano-formulations high fluorescence quantum yield, fluorescence brightness, absorption coefficient, and good fluorescence and photothermal properties, making them of great application potential in chemical and biological sensing, biomedical imaging, and optical diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the fields of functional fluorescent dyes and biomedical technology, specifically relating to a near-infrared squaricine dye with ultra-high fluorescence brightness, its preparation method, and its application. Background Technology

[0002] Near-infrared II fluorescence imaging (NIIR II) is characterized by low absorption and low scattering rates in biological tissues, resulting in stronger tissue penetration and significantly increased imaging depth. Furthermore, due to the limited autofluorescence of biological tissues in this band, the imaging exhibits a higher signal-to-noise ratio and sensitivity. NIIR II fluorescence imaging also offers advantages such as non-invasiveness, biocompatibility, and selective irradiation, making it a promising clinical diagnostic and therapeutic tool.

[0003] In fluorescence imaging, higher brightness helps improve the signal-to-noise ratio, making the target structure more clearly visible. Sufficient brightness also excites the photosensitizer or generates enough signal to ensure adequate detection and identification of the target region. Currently, in organic near-infrared small molecule dyes, besides the wavelength, the fluorescence brightness of the dye molecule plays a crucial role in the clinical application of organic dyes in optometry. Therefore, developing a formulation with high fluorescence quantum yield, fluorescence brightness, absorption coefficient, and good fluorescence and photothermal properties has great application potential. Summary of the Invention

[0004] In view of the above-mentioned shortcomings mentioned in the background art, the purpose of this invention is to provide a near-infrared squartzine dye with ultra-high fluorescence brightness, its preparation method and application.

[0005] To achieve the above objectives, the first objective of this invention is to provide a near-infrared squartzine dye with ultra-high fluorescence brightness, the chemical molecular structure of which is shown below:

[0006]

[0007] Wherein, R is an alkyl chain with 3 to 12 carbon atoms;

[0008] M is a coordinating anion, ClO4. - Cl - ,Br - , I - Any one of them.

[0009] Furthermore, in the above technical solution, the chemical molecular structure of the near-infrared cyanine dye is as follows:

[0010] .

[0011] The second objective of this invention is to provide a method for preparing a near-infrared squaric acid cyanine dye with ultra-high fluorescence brightness. The method for preparing the near-infrared squaric acid cyanine dye is as follows: a 1,8-naphthoylimide molecule having a side chain quaternary ammonium salt group is coupled with a squaric acid molecule to obtain the near-infrared squaric acid cyanine dye.

[0012] Furthermore, in the above technical solution, the structure of the 1,8-naphtholide molecule with side-chain quaternary ammonium salt groups is shown in the following formula:

[0013] In this context, R is an alkyl chain with 3 to 12 carbon atoms; M is a coordinating anion, which is ClO4. - Cl - ,Br - , I - any one of them;

[0014] The structure of the squaric acid molecule is shown in the following formula:

[0015] .

[0016] Furthermore, in the above technical solution, the preparation method of the near-infrared squaric acid cyanine dye is specifically as follows: 1,8-naphthoylimide molecules with side-chain quaternary ammonium salt groups and squaric acid molecules are added to a Dean-Stark apparatus containing a n-butanol / toluene solution and refluxed for 2 h. After the reaction is complete, the solvent is removed by vacuum evaporation, and the crude product is purified by reverse silica gel column chromatography to obtain the target product (SQNMe). The synthetic route is shown in the following formula:

[0017] .

[0018] Furthermore, in the above technical solution, the molar ratio of the 1,8-naphthoimide molecule with the side chain quaternary ammonium salt group to the squaric acid molecule is 2~2.2:1; the volume ratio of the n-butanol and toluene is 1:1; and the eluent used for column chromatography is a mixture of methanol, water and trifluoroacetic acid in a volume ratio of 1~2:10:0.1.

[0019] Furthermore, in the above technical solution, the 1,8-naphtholide molecule with a side-chain quaternary ammonium salt group is prepared by the following method:

[0020] (1) Under nitrogen protection, 1,8-naphthoimide and dibromoalkane were added to a 100 mL round-bottom flask, followed by potassium carbonate and acetonitrile. The reaction was heated to 90 °C and refluxed for 12 h. After the reaction was completed, acetonitrile was removed by rotary evaporation, and the organic phase was extracted with dichloromethane / water. The organic phase was dried, the solvent was evaporated under reduced pressure, and the organic phase was purified by silica gel column chromatography with petroleum ether and ethyl acetate as eluents to obtain oily compound 1.

[0021] (2) The above-mentioned oily tetrahydrofuran solution and trimethylamine solution of compound 1 were added dropwise and stirred under nitrogen protection. The mixture was stirred at room temperature for 48 h. The mixture and residue after vacuum filtration were washed with ethyl acetate and n-hexane to obtain yellow solid compound 2.

[0022] (3) Compound 2 was mixed with methyl magnesium chloride solution in anhydrous tetrahydrofuran solution at 0°C, stirred at 60°C for 2 h, cooled to 0°C, and then H2O and dilute hydrochloric acid were added. The mixture was stirred for 15 min, followed by the addition of potassium iodide solution. The resulting mixture was extracted, filtered, and the solvent was evaporated under rotary evaporation to obtain black oily liquid compound 3. The specific reaction formula is shown below:

[0023] .

[0024] Further, in the above technical solution, in step (1), the molar ratio of 1,8-naphthoimide, dibromoalkane and potassium carbonate is 1:10:1.5, and the eluent for silica gel column chromatography is a mixture of petroleum ether and ethyl acetate in a volume ratio of 100~50:1; in step (2), the tetrahydrofuran solution of trimethylamine is 2 mol / L, and the molar ratio of compound 1 and trimethylamine is 1:2~4; in step (3), the concentration of the tetrahydrofuran solution of methyl magnesium chloride is 3 mol / L, and the molar ratio of compound 2, methyl magnesium chloride and potassium iodide is 1:3:1~3.

[0025] A third objective of this invention is to provide a nano-formulation comprising the near-infrared oxocyanine dye described in any of the preceding claims.

[0026] The fourth objective of this invention is to provide a method for preparing nano-formulations, comprising the following steps:

[0027] SQNMe was dissolved in a methanol solution, and the SQNMe methanol solution was added dropwise to mPEG under ultrasonic treatment. 2k The SQNMe nano-formulation was obtained by vacuum drying in an aqueous solution of DSPE ammonium salt and then sonicating in a cell disruptor at 25°C for 20 min to obtain a clear SQNMe nano-formulation. The nano-formulation was then dialyzed in water for 24 h using a dialysis bag and filtered through a 220 nm pore size filter membrane for later use.

[0028] Specifically, in the above technical solution, methanol and mPEG 2k The volume ratio of the aqueous solution of -DSPE ammonium salt is 1:3~6; SQNMe and mPEG 2k The molar ratio of -DSPE ammonium salt is 1:20~100; the molecular weight cutoff of the dialysis bag is 100kDa.

[0029] The fifth objective of this invention is to provide an application of the above-mentioned nano-formulation in bioimaging products.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention designs and synthesizes a novel squaric acid cyanine dye. The DAD-type squaric acid cyanine dye is formed by coupling a quaternary ammonium salt group to a 1,8-naphthoylimide molecule with squaric acid molecules. This molecule exhibits good water solubility. The quaternary ammonium salt group anchors the molecule itself through electrostatic interaction with the squaric acid cyanine dye parent molecule, significantly enhancing the nonradiative transition rate. Simultaneously, electrostatic self-assembly with DSPE-PEG further anchors the molecule within liposome nanoparticles to reduce molecular vibration. This results in nanoformulations with high fluorescence quantum yield, fluorescence intensity, absorbance coefficient, and excellent fluorescence and photothermal properties. The fluorescence intensity is 58 times higher than that of unco-assembled squaric acid cyanine dye at the same concentration in water, demonstrating significant application potential in chemical and biological sensing, biomedical imaging, and optical diagnostics. The compound is simple to synthesize and the reaction is easily controlled. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The 1H NMR spectrum of the ultrabright near-infrared squaric acid cyanine dye in Example 1 of this invention;

[0034] Figure 2 This is the high-resolution mass spectrometry characterization spectrum of the ultra-bright near-infrared squaric acid cyanine dye in Example 1 of the present invention;

[0035] Figure 3 The fluorescence absorption and emission spectra of the ultra-bright near-infrared squaric acid cyanine dye in Example 1 of this invention are shown.

[0036] Figure 4 The ultraviolet-visible-near-infrared absorption spectra and fluorescence spectra of the ultra-bright near-infrared squaric acid cyanine dye in different solvents in Example 2 of the present invention are shown.

[0037] Figure 5 Transmission electron microscope images and particle size analysis diagrams of the ultra-bright near-infrared squaric acid cyanine dye nanoformation obtained in Example 2 of this invention;

[0038] Figure 6 This is a graph showing the enhanced fluorescence intensity of the ultra-bright near-infrared squaric acid cyanine dye nano-formulation obtained in Example 2 of this invention;

[0039] Figure 7 These are near-infrared imaging camera images of the ultra-bright near-infrared cyanine dye nano-formulation obtained in Example 3 of this invention.

[0040] Figure 8 Examples of near-infrared II nano-formulations used in Example 3 of this invention are used to obtain in vivo vascular imaging images and tumor imaging images of mice. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials used in the following examples are all commercially available products and can be purchased from the market.

[0042] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0043] The raw materials involved in the various embodiments of the present invention are either commercially available products or can be prepared according to existing methods.

[0044] The specific content of the present invention will be further explained and described below with reference to the embodiments.

[0045] The structure of the ultrabright near-infrared squaric acid cyanine dye was characterized and confirmed using 1H NMR and high-resolution mass spectrometry. The instrument used for detection was an AVANCE III HD 400MHz spectrometer (Bruker, Germany), and high-resolution mass spectrometry (HRMS) was performed using a UHPLC30A-Triple TOF 5600+ instrument (SCIEX, Japan). UV-Vis-NIR absorption spectra were measured using a Lambda 750 spectrophotometer (PerkinElmer, USA). Photoluminescence (PL) spectra were measured using an FLS1000 fluorescence spectrophotometer (Edinburgh, UK). All experiments involving fluorescence imaging were performed using MARS (ARTEMISINTELLIGENT imaging, China).

[0046] Example 1

[0047] A near-infrared squaring cyanine dye with ultra-high fluorescence brightness, SQNMe:

[0048] The molecular structure of the ultra-bright square borate dye SQNMe in this embodiment is shown in the figure below:

[0049] The specific synthesis route is as follows:

[0050]

[0051] The preparation method of this embodiment includes the following steps:

[0052] (1) 1,8-naphthoimide (benzo[cd]indol-2(1H)-one, 1.69 g, 10 mmol) and potassium carbonate (K2CO3, 32.07 g, 15 mmol) were added to acetonitrile (50 mL), stirred, and then 1,3-dibromopropane (20.1 g, 100 mmol) was added dropwise. The mixture was stirred at 85 °C for 5 h. The mixture was then cooled to room temperature, filtered to remove potassium carbonate, and the solvent was removed under reduced pressure. The crude oil was purified by silica gel column chromatography using petroleum ether / ethyl acetate (50 / 1) as the eluent to obtain a yellow oily compound 1 (1.80 g, 62%).

[0053] (2) A solution of compound 1 (1.47 g, 5 mmol) in tetrahydrofuran (THF, 30 mL) was gradually added to a trimethylamine solution (5 mL, 10 mmol / L) under N2 protection. The mixture was stirred at room temperature for 48 h. After vacuum filtration, the mixture and residue were washed with ethyl acetate and n-hexane to give a yellow solid compound 2 (1.60 g, 92%).

[0054] (3) 3.0 mL of 3.0 mol / L tetrahydrofuran solution and 9.0 mmol of methyl magnesium chloride (CH3MgCl) were added to an anhydrous tetrahydrofuran (20 mL) solution of compound 2 (1.05 g, 3.0 mmol) at 0 °C. The mixture was stirred at 60 °C for 2 h, cooled to 0 °C, and then H2O (0.5 mL) and dilute hydrochloric acid (15% aqueous solution, 5.0 mL) were added. The mixture was stirred for 15 min, followed by the addition of potassium iodide solution (3 mol / L, 3.0 mL). The resulting mixture was extracted, filtered, and the solvent was evaporated under rotary evaporation to obtain a black oily liquid compound 3. The crude product obtained was used in subsequent steps without further purification.

[0055] (4) A mixture of compound 3 (3.0 mmol) and squaric acid (3,4-dihydroxycyclobut-3-ene-1,2-dione, 0.17 mg, 1.5 mmol) was added to a butanol-toluene solution (v:v = 1:1, 20 mL). The mixture was then heated at 115 °C for 2 h under reflux in a Dean-Stark apparatus. After the reaction was complete, the mixture was filtered, and the solid residue was washed with acetone. Subsequently, the mixture was analyzed by reversed-phase chromatography (C10). 18 The mixture was purified by adding 10-20% MeOH in H2O + 0.1% TFA, and the black solid SQNMe (398 mg, 34%) was separated.

[0056] The obtained ultrabright near-infrared squaring cyanine dye SQNMe was characterized by 1H NMR and high-resolution mass spectrometry, and the results are as follows: Figure 1 and Figure 2 As shown. The obtained ultra-bright near-infrared cyanine dye SQNMe was subjected to spectral analysis, and the resulting absorption and emission spectra are shown below. Figure 3 As shown in the figure. These results indicate that the synthesized compound structure is the designed ultrabright near-infrared squaricine dye SQNMe, and it exhibits fluorescence emission in the near-infrared II region.

[0057] Example 2

[0058] Superb near-infrared cyanine dye SQNMe and mPEG 2k - A method for preparing nano-formulations by co-assembly of DSPE ammonium salts includes the following steps:

[0059] SQNMe (0.86 mg) was dissolved in 1 mL of methanol and added dropwise to 3 mL of mPEG-DSPE ammonium salt (13.33 mg / mL) solution under sonication. After addition, the dispersion was exposed to ultrasound at 25 °C for 20 min, followed by sonication for another 15 min using an ultrasonic cell disruptor. The methanol solution was removed by rotary evaporation, and the solution was then carefully filtered and purified using a 220 nm syringe filter. Subsequently, the solution was dialyzed for 2 days using a dialysis bag with a molecular weight of 100 kDa. The solution collected from the dialysis bag after 2 days was identified as a nano-formulation of SQNMe and stored at 4 °C for subsequent use.

[0060] The absorption spectra of the nano-formulation were measured using a UV-Vis-NIR absorption spectrometer, and the photoluminescence spectra were measured using a steady-state fluorescence spectrometer. The results are as follows: Figure 4 As shown in the figure. Transmission electron microscopy and dynamic light scattering particle size analyzer were used to characterize the morphology and size of the nanocomposite, and the results are as follows. Figure 5 As shown, the obtained nano-formulation is spherical with a particle size of approximately 85 nm.

[0061] An aqueous solution of SQNMe, a dimethyl sulfoxide (DMSO) solution, and a nano-formulation (SQNMe) of the same concentration were placed under 808 nm laser irradiation, and a CCD camera recorded the results. Figure 6 As shown, the fluorescence intensity was analyzed using software as follows: Figure 6 As shown. The results show that the ultra-bright near-infrared cyanine dye SQNMe and mPEG... 2k The nanoformulations prepared by co-assembly of DSPE ammonium salt exhibited a 58-fold increase in fluorescence intensity compared to the aqueous solution of SQNMe, demonstrating a significant fluorescence enhancement effect. This indicates that the prepared nanoformulations possess high fluorescence intensity under 808 nm laser irradiation, providing a good foundation for imaging.

[0062] Example 3

[0063] Superb near-infrared cyanine dye SQNMe and mPEG 2k Nanoformations prepared by co-assembly of DSPE ammonium salt were used for near-infrared II fluorescence in vivo vascular imaging and tumor imaging in mice.

[0064] Balb / c-Nude mice aged 6-8 weeks were used. The prepared nano-formulation was injected into the mice via the tail vein. The mice were then exposed to 808 nm laser irradiation, and images were acquired by a CCD camera under a 1000 nm long-pass filter.

[0065] Balb / c-Nude tumor-bearing mice aged 6-8 weeks were used. The prepared nano-formulation was injected into the mice via the tail vein. The mice were exposed to 808nm laser irradiation at 0.1, 4, 8, 12, 24, and 36 hours. Images were then acquired by a CCD camera under a 1000nm long-pass filter.

[0066] Fluorescence images acquired by a CCD camera (see) Figure 7 The images clearly demonstrate how, after tail vein injection, the nanoparticles are distributed throughout the mouse's blood vessels as they circulate in the bloodstream. Due to the high penetrability of near-infrared II fluorescence, the images reveal bright outlines of the blood vessels, penetrating the mouse's tissues and skin.

[0067] Further observation Figure 8 We can observe that after tail vein injection, the nanoparticles actively participate in blood circulation and significantly accumulate in the tumor. The peak of accumulation occurs approximately 12 hours post-injection, at which point imaging of the tumor region reaches its optimal state, and this accumulation can persist for about 36 hours. This demonstrates the dynamic behavior of the nanoparticles in vivo and their long-term retention in tumor tissue, providing strong imaging support for further research and applications.

[0068] In summary, the near-infrared cyanine dye of the present invention exhibits good water solubility. After introducing charged groups, the non-radiative transition rate of the molecule is significantly enhanced, and this is further improved by reacting with mPEG. 2k The nanoformulations obtained by co-assembling DSPE ammonium salt exhibit strong fluorescence emission, with a fluorescence intensity that is 58 times greater than that of unco-assembled azircetin dyes of the same concentration in water. This can provide a basis for subsequent chemical and biological sensing, biomedical imaging, and optical diagnosis and treatment.

[0069] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A near-infrared squartzine dye, characterized in that, The chemical molecular structure of the near-infrared squaring cyanine dye is shown below: 。 2. The method for preparing a near-infrared oxocyanine dye as described in claim 1, characterized in that, The near-infrared squaric acid cyanine dye is prepared by coupling a 1,8-naphthoylene imide molecule with a side-chain quaternary ammonium salt group to a squaric acid molecule to obtain the near-infrared squaric acid cyanine dye; the structure of the 1,8-naphthoylene imide molecule with the side-chain quaternary ammonium salt group is shown in the following formula: Where R is an alkyl chain with 3 carbon atoms; M is a coordinating anion, which is Cl. - ; The structure of the squaric acid molecule is shown in the following formula: 。 3. The method for preparing a near-infrared oxocyanine dye according to claim 2, characterized in that, The preparation method of the near-infrared squaric acid cyanine dye is as follows: 1,8-naphthoimide molecules with side-chain quaternary ammonium salt groups and squaric acid molecules are added to a Dean-Stark apparatus containing n-butanol / toluene solution and refluxed for 2 h. After the reaction is completed, the solvent is removed by vacuum evaporation, and the crude product is purified by reverse silica gel column chromatography to obtain the target product.

4. The method for preparing a near-infrared cyanine dye according to claim 3, characterized in that, The molar ratio of the 1,8-naphthoimide molecule with the side chain quaternary ammonium salt group to the squaric acid molecule is 2~2.2:1; the volume ratio of the n-butanol to toluene is 1:1; the eluent used for column chromatography is a mixture of methanol, water and trifluoroacetic acid in a volume ratio of 1~2:10:0.

1.

5. The method for preparing a near-infrared cyanine dye according to claim 2, characterized in that, The 1,8-naphtholide molecule with side-chain quaternary ammonium salt groups was prepared by the following method: (1) Under nitrogen protection, 1,8-naphthoimide and 1,3-dibromopropane were added to a 100 mL round-bottom flask, followed by potassium carbonate and acetonitrile. The reaction was heated to 90 °C and refluxed for 12 h. After the reaction was completed, the acetonitrile was removed by rotary evaporation, and the organic phase was extracted with dichloromethane / water. The organic phase was dried, the solvent was evaporated under reduced pressure, and the organic phase was purified by silica gel column chromatography with petroleum ether and ethyl acetate as eluents to obtain an oily compound 1, the chemical molecular structure of which is shown in Formula 1. ; (2) The above-mentioned oily tetrahydrofuran solution and trimethylamine solution of compound 1 were added dropwise and stirred under nitrogen protection. The mixture was stirred at room temperature for 48 h. The mixture and residue after vacuum filtration were washed with ethyl acetate and n-hexane to obtain yellow solid compound 2, whose chemical molecular structure is shown in Formula 2: ; (3) The yellow solid compound 2 was mixed with methyl magnesium chloride solution in anhydrous tetrahydrofuran solution at 0°C and stirred at 60°C for 2 h. After cooling to 0°C, H2O and dilute hydrochloric acid were added and stirred for 15 min. Then potassium iodide solution was added. The resulting mixture was extracted, filtered, and the solvent was evaporated under rotation to obtain a 1,8-naphthoimide molecule with a side chain quaternary ammonium salt group, which is a black oily liquid compound 3 with the molecular structure of formula 3. .

6. A method for preparing a near-infrared cyanine dye according to claim 5, characterized in that, In step (1), the molar ratio of 1,8-naphthoimide, 1,3-dibromopropane and potassium carbonate is 1:10:1.5, and the eluent for silica gel column chromatography is a mixture of petroleum ether and ethyl acetate in a volume ratio of 100~50:1; in step (2), the tetrahydrofuran solution of trimethylamine is 2 mol / L, and the molar ratio of oily compound 1 and trimethylamine is 1:2~4; in step (3), the tetrahydrofuran solution of methyl magnesium chloride is 3 mol / L, and the molar ratio of yellow solid compound 2, methyl magnesium chloride and potassium iodide is 1:3:1~3.

7. A nano-formulation, characterized in that, It contains the near-infrared oxocyanine dye as described in claim 1.

8. The use of a nanoformulation as described in claim 7 in the preparation of bioimaging articles.

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